Muhammad Mahran Aslam
Plants regenerated from cell cultures may exhibit phenotypes differing from their parent plants, sometimes at quite high frequencies. If these are heritable and affecting desirable agronomic traits, such "somaclonal variation" can be incorporated into breeding programs. However, finding of specific valuable traits by this method is largely left to chance and hence inefficient. Rather than relying on this undirected process, selection in vitro targets specific traits by subjecting large populations of cultured cells to the action of a selective agent in the Petri dish. For purposes of disease resistance, this selection can be provided by pathogens, or isolated pathotoxins that are known to have a role in pathogenesis. The selection will only allow those cells to survive and proliferate that are resistant to the challenge. Selection of cells also plays an important role in genetic engineering, where special marker genes are used to select for transgenic cells.
Somatic hybrid plants are plants derived from the fusion of somatic cells. Cell fusion was developed after the successful culture of a large number of plant cells stripped of their cell walls. The resulting cells without walls are referred to as protoplasts. Since also protoplasts from phylogenetically unrelated species can be fused, attempts have been made to overcome sexual incompatibility using protoplast fusion. In most cases, these attempts failed because growth and division of the fused cells did not take place when only distantly related cells were fused. Successful fusions between sexually incompatible petunia species and between potatoes and tomatoes did not lead to economically interesting products, but important contributions to the understanding of cell wall regeneration and other mechanisms were achieved.
Genetic engineering is a term used for the directed manipulation of genes, i. e. the transfer of genes between organisms or changes in the sequence of a gene. Closely related to this field are methods which use genes or specific sequences for the identification of traits and other analytical purposes. In plant breeding, the most important and already widely used method of this kind is Restriction Fragment Length Polymorphism (RFLP)
In conventional breeding, the pool of available genes and the traits they code for is limited due to sexual incompatibility to other lines of the crop in question and to their wild relatives. This restriction can be overcome by using the methods of genetic engineering, which in principle allow introducing valuable traits coded for by specific genes of any organism (other plants, bacteria, fungi, animals, viruses) into the genome of any plant. The first gene transfer experiments with plants took place in the early 1980s. Normally, transgenes are inserted into the nuclear genome of a plant cell. Recently it has become possible to introduce genes into the genome of chloroplasts and other plastids (small organelles of plant cells which possess a separate genome).
Transgenic plants have been obtained using Agrobacterium-mediated DNA-transfer and direct DNA-transfer, the latter including methods such as particle bombardment, electroporation and polyethylenglycol permeabilisation. The majority of plants have been transformed using Agrobacterium mediated transformation.
Particle Bombardment also referred to as biolistic transformation (from biological ballistics) involves coating biologically active DNA onto small tungsten or gold particles (1-5 µm in diameter) and accelerating them into plant tissue at high velocity. The particles penetrate the plant cell wall and lodge themselves within the cell where the DNA is liberated resulting in transformation of the individual plant cell in an explant. This technique is generally less efficient than Agrobacterium-mediated transformation, but has nevertheless been particularly useful in several plant species, most notably in cereal crops. The introduction of DNA into organized, morphogenic tissues such as seeds, embryos or meristems has enabled the successful transformation and regeneration of rice, wheat, soybean and maize, thus demonstrating the enormous potential of this method.
Electroporation is a process whereby very short pulses of electricity are used to reversibly permeabilize lipid bilayers of plant cell membranes. The electrical discharge enables the diffusion of macromolecules such as DNA through an otherwise impermeable plasma membrane. Because the plant cell wall will not allow the efficient diffusion of many transgene constructs, protoplasts (cells without cell walls) must be prepared. This requirement presents a major obstacle for many applications as protocols making possible the regeneration of protoplasts into complete plants do not exist for many species.
In most cases, the introduction of a gene into the plant genome will only have an effect on the plant if the transgene is expressed, i. e. transcribed into mRNA and translated into a protein. A promoter is a sequence of nucleic acids where the RNA polymerase (a complex enzyme synthesizing the mRNA transcript) attaches to the DNA template. The nature of the promoter defines (together with other expression-regulating elements), under which conditions and with which intensity a gene will be transcribed. The promoter of the 35S gene of cauliflower mosaic virus is used very frequently in plant genetic engineering. This promoter confers high-level expression of exogenous genes in most cell types from virtually all species tested. As it is often advantageous to express a transgene only in certain tissues or quantities or at certain times, a number of other promoters are available, e.g. promoters inducing gene expression after wounding or during fruit ripening only.
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